Electrical sensor systems for analyzing analytes in biological samples are widely used. For example, analytes such as glucose level, cholesterol level or uric in a sample such as blood may be analysed. In general, such electrochemical measuring systems include a test strip and a measuring meter. In particular, test strips are provided as single use, disposable strips for easy home use.
In particular, electrochemical sensors using enzymatic amperometric methods are well known. The sensors of such systems have electrodes coated with a reagent including enzymes. The electrodes are used to sense an electrochemical current produced by a reaction between the reagent and the analyte or the analytes in a test sample. The enzyme is used for a unique, well specified reaction with a specific analyte in the test sample. This specific reaction reduces the interference with other analytes. A reagent with a specific cholesterol enzyme may e.g. be used to test the cholesterol level in a sample. A reagent with a glucose oxidase enzyme may be used to measure the glucose level in a blood sample. The glucose oxidase does not react with cholesterol. Neither does it react with other sugars in the blood sample. The use of glucose oxidase e.g. typically leads to a 99% unique selection of glucose within the sample. Methods based on the use of enzyme are leading to most accurate measurement results.
Currently, a plurality of generations of amperometric methods have been developed for analysis of the glucose level in a blood sample.
In a first generation of glucose analysis, the reagent covering the sensor includes glucose oxidase enzymes (hereinafter referred to as GOD). The mechanism of the reaction between the glucose in the sample and the enzyme and subsequent detection are as follows:Glucose+GOD(ox)→Gluconic acid+GOD(red)  (1)O2+GOD(red)→H2O2+GOD(OX)  (2)H2O2(Apply Voltage)→O2+2H++2e−  (3)
GOD has two forms. In equation (1), GOD is present in its natural, oxidised form GOD(ox) before its reaction with glucose. In the reaction with glucose, GOD is changed to the reduced form GOD(red).
After reaction with oxygen O2, GOD(ox) is returned to the oxidised form GOD(ox) (see equation (2)). In this reaction, hydrogen peroxide H2O2 is created. GOD is oxidised again, will be still present in the reagent and can be reused for a reaction according to equation (1). The amount of produced H2O2 is proportional to the level of glucose in the sample, provided there is a sufficient amount of oxygen O2 present. If the amount of H2O2 can be determined, the glucose level can be determined. In order to measure the amount of H2O2 produced, a predetermined voltage is applied to the electrodes such as to oxidise H2O2 (see equation (3)). By oxidation, two electrons e− will be delivered and H2O2 will be oxidised to oxygen O2 and two hydrogen 2H+. Oxygen O2 again can be used in the reaction according to equation (2). The electrons generated at the electrodes provide a current. This sensing current is proportional to the glucose level in the sample.
This first generation glucose analysis method has some drawbacks which are in particular due to an insufficient amount of oxygen O2 initially present before the start of the reaction process. Especially in home use applications based on disposable test strips of glucose sensors, reagents of a dry type are used in view of long storage time. In such sensors, initial oxygen is provided by the blood sample only. While there is some oxygen inside a fresh blood sample, this may be an insufficient amount especially for high concentrations of glucose. This may lead to measurement errors. Furthermore, red blood cells within the sample may continuously consume oxygen so that oxygen will be exhausted. An insufficient amount of oxygen may lead to a reading error. This phenomenon is known as the oxygen dependence problem.
In order to solve the oxygen dependence problem, in a second generation of glucose sensor, electron carrying mediators are used in the reagent. Typically, potassium ferrocyanide is used as a mediator. In case of strips using such mediators, the reaction is as follows:Glucose+GOD(ox)→Gluconic acid+GOD(red)  (4)GOD(red)+2M(ox)→2M(red)+GOD(OX)+2H+  (5)2M(red)(Apply Voltage)→2M(ox)+2e−  (6)
In the above equations, M(ox) represents the oxidised and M(red) represents the reduced form of the mediator. The reaction as shown in equations (4) to (6) are such that during a reaction cycle a sensing current is produced which depends on the concentration of the glucose in the blood sample. The whole reaction process is thus basically independent of the amount of oxygen in the sample. However, the effect from oxygen during the mediator reaction cannot be fully avoided. Equation (4) is basically identical to equation (1) shown above. Oxygen O2 present in the initial blood will react with the reduced form of GOD and thereby also create hydrogen peroxide H2O2 as shown in equation (2). If a predetermined voltage is applied to the electrodes in order to oxidise the mediator in reaction (6), the same voltage will lead to oxidation of H2O2. Electrons based on oxidation of H2O2 (as shown in equation (3)) will thus be generated at the electrodes. In consequence, the sensing current is constituted by two parts. A first part is based on oxidation of the mediator in the reduced form (see equation (6)). A second part of the current is based on the reaction (3) shown above. Because of the unstable amount of oxygen in the blood sample, this secondary current is unstable. The measurement result is thus unstable. This phenomenon is also called the oxygen interference.
There have been several proposals in order to overcome the oxygen interference in the second generation of glucose sensors. One solution is to replace the glucose oxidase enzyme with glucose dehydrogenase (GDH). Because GDH does not react with oxygen such as to create H2O2, there is no oxygen interference. However, there are some drawbacks with respect to the use of GDH. In particular, the selectivity with respect to glucose is only about 80 to 90%. The GDH does not only react with glucose but also with other sugars present in the blood such as maltose. While the use of GDH reduces the oxygen interference problem, it creates another interference problem, leading to erroneous readings of the glucose level.
In order to determine the concentration of the analyte in the sample, the sensing current is measured. The sensing current is called the Cottrell current, according to the following equation:i(t)=K·n·F·A·C·D0.5·t−0.5 Where: i is an instant value of the sensing current;                K is a constant;        n is the transferred number of electrons (For example, n is 2 in the equation (6));        F is the Faraday constant;        A is the surface area of the working electrode;        C is the concentration of the analyte in the sample;        D is the diffusion coefficient of the reagent;        t is a specified time after a predefined voltage has been applied to the electrodes.        
The concentration C of the analyte shall be determined. This concentration is proportional to the sensing current i. Because the sensing current is also proportional to the surface area A of the working electrode, a precisely defined surface area of the working electrode of the test strip is a key factor in view of an accurate measurement.
Furthermore, as shown in the Cottrell Equation, the time dependent value of the sensing current decreases with the square root of the duration after the time when the predefined voltage has been applied to the electrodes. The control of the point in time when a voltage is applied to the electrodes and when the Cottrell current is determined is a further important factor in view of accurate measurements.
Examples of such sensors and test meters are e.g. disclosed in U.S. Pat. No. 5,266,179, U.S. Pat. No. 5,366,609 or in EP 1 272 833.
The operation principle of the measuring meters disclosed in these documents is generally the same. First, a test strip is inserted into the measuring meter. A proper insertion of the test strip within the meter is detected by mechanical and/or electrical switches or contacts. Once a test strip is properly inserted into the measuring meter, the user is asked to provide a sample, typically a drop of blood. The sample of blood is then fed to a reaction zone on the test strip. The reaction zone of the test strip is provided with at least two electrodes which are covered by the reagent.
In order to detect presence of a sample in the reaction zone, secondly, a voltage is applied to the electrodes. The resistance of the reagent between the electrodes without the presence of a sample is high. As soon as a sample is present in the reaction region, the resistance between the electrodes is reduced. Reduction of the resistance leads to flow of a current which may be detected as an indication of the presence of a sample.
A further drawback of known measuring meters is related to such detection of sample presence. In a sample detection period, a voltage is applied to the electrodes for verifying whether or not a sample is present. This voltage, however, leads to consumption of the current (i.e. consumption of electrodes) which is produced as a result of the reaction between the reagent and the testing sample. The current is related to the concentration of the analyte in the sample. The consumption of current during sample pressure detection leads to measurement errors. This problem is particularly relevant if the sample of the analyte is relatively small or if the detection time in the measurement system is relatively short. Especially for home use measuring systems, the volume of the sample and the detecting time of the measuring meters has been recently reduced in order to increase the usability and the user friendliness of such test strips. Typically, the volume of blood samples has been reduced from originally 10 mL to actually around 0.3 mL. The total detecting time has been reduced from originally about 60 seconds to actually about 5 seconds.
Once a sufficient amount of sample is present in the reaction zone, in a second step mixing of the sample with the reagent is allowed for a certain period of time. This period of time is also called incubation time. After completion of incubation, in a third step, the measurement starts.
Another problem of known devices is related to the incubation time. Incubation time is used in order to allow mixing and melting of the sample with the reagent. A certain time is needed for completion of this mixing and melting. The completion of the melting is affected by parameters such as the ambient temperature or sample blood conditions of the user. The melting is e.g. slow at low ambient temperature or with patients having a high fat proportion within the blood. If measurements are made before melting has been completed, an unstable measuring current will result. Consequently, currently, a sufficient incubation time must be selected such as to be suitable for the longest melting conditions in order to guarantee a precise and accurate measurement under all circumstances.
If a voltage is applied during incubation, current starts to be consumed during the incubation period. Because of different melting conditions, the amount of consumption is unstable. Therefore, again, measurement errors would be caused if a voltage is applied.
Operation and drawbacks of such prior art meters will be explained in more detail in FIGS. 3A to 3C.
Some prior art documents such as U.S. Pat. No. 5,108,564 suggest to make incubation in an open circuit. This measurement principle has, however, also certain drawbacks. During the incubation time, small oxygen bubbles are created in the sample if no voltage is applied. Generation of oxygen bubbles is due to the following process. As mentioned above, the use of electron mediators solves the oxygen dependency problem in context with first generation strips. However, as mentioned above, there is still a problem of oxygen interference. One solution to the oxygen interference problem is to select a specific mediator material only requiring a low voltage such as to oxidise the mediator as shown in equation (6). If the voltage necessary to oxidise the mediator is insufficient to oxidise H2O2 as shown in equation (3), the oxygen interference can be reduced to an extent at which it can be ignored. In particular, the ratio between primary and secondary current can be increased to 20 to 1 or even above 150 to 1 when the predefined voltage is applied to the electrodes during the measurement time period.
No voltage is applied during the incubation time according to the conventional measuring meters. If no voltage is applied, the reaction processes of the equations (3) and (6) will be stopped during incubation time. At the beginning of incubation time, glucose will react with the oxidised form of the glucose oxidase GOD(ox), thereby reducing GOD to the reduced form GOD(RED). Because the reduced form of the mediator cannot be oxidised into its oxidised form M(ox) without application of the predefined voltage to the electrodes, GOD(RED) will react with both the mediator and oxygen in accordance with equations (2) and (5). The reaction according to equation (5) will be stopped once the mediator present in the reagent has been brought to the reduced form M(RED).
In parallel, initial oxygen within the fresh blood will create H2O2 in accordance with equation (2). H2O2 cannot be oxidised to O2 and 2H+ and electrons in accordance with equation (3) if no voltage is applied to the electrodes. However, H2O2 will naturally dissolve into oxygen O2 and water H2O under the effect of temperature, as shown in equation (7). Furthermore, if no voltage is applied, a metal electrode catalyzes the dissolution of H2O2 to O2 and H2O in accordance with the following equation (8):H2O2(under temperature)→O2(gas)↑+H2O  (7)H2O2(catalysis by metal)→O2(gas)↑+H2O  (8)
Initially, oxygen O2 is uniformly distributed within the fresh sample. Because the reaction in accordance with equation (8) above is catalysed by the metal electrodes, O2 will accumulate in the form of small bubbles in the area of the electrode surface. Because usually a capillary channel will be formed neighbouring the electrodes in order to automatically suck the sample into the reaction zone, oxygen bubbles attached to the electrode can-not easily disappear. Consequently, the effective working area of the electrode is reduced by accumulation of oxygen bubbles. As a consequence, the measuring current after incubation time will be affected. Especially if small sample volumes are used, the effect of oxygen bubbles attached to the electrode surface may become relatively important such that measurement errors may become considerable. In particular, if electrodes made of a noble metal are used, formation of such oxygen bubbles may become important.
In EP 1 272 833, it has been suggested to apply a voltage during incubation time or during sample detection time which is, however, relatively low. This solution does not solve the problem of creation of oxygen bubbles.
It is therefore an object of the present invention to overcome the drawbacks of the prior art, in particular to provide a method for operating a measuring meter which allows for very accurate analyte measurements even if the volume of sample used is relatively small and/or the measuring time is short.
It is in particular an object of the present invention to provide a solution to the competition between the reaction involving oxygen in accordance with equation (2) and a reaction involving a mediator in accordance with equation (5) in second generation test strips, i.e. to reduce the oxygen interference. It is a further object of the present invention to increase the accuracy of measurements by maintaining a stable and precise surface area of the working electrode. It is a further object of the invention to provide a measurement strip and a meter which allow for a precise timing of incubation time and measurements.
According to the present invention, these and other objects are solved with a method for operating a measuring meter and with a measuring meter according to the features of the characterising portion of the independent patent claims.